Protein C is a zymogen, or precursor, of a serine protease that plays an important role in the regulation of blood coagulation and in the generation of fibrinolytic activity in vivo. It is synthesized in the liver as a single-chain polypeptide that undergoes considerable processing to give rise to a two-chain molecule comprising heavy (Mr=40,000) and light (Mr=21,000) chains held together by a disulfide bond. The circulating two-chain intermediate is converted to the biologically active form of the molecule, known as "activated protein C" (APC), by the thrombin-mediated cleavage of a 12-residue peptide (also know as the activation peptide) from the amino-terminus of the heavy chain. The cleavage reaction is augmented in vivo by thrombomodulin, an endothelial cell co-factor (Esmon and Owen, Proc. Natl. Acad. Sci. USA 78:2249-2252, 1981).
Protein C is a vitamin K-dependent glycoprotein that contains approximately nine residues of gamma-carboxyglutamic acid (Gla) and one equivalent of beta-hydroxyaspartic acid, which are formed by post-translational modifications of glutamic acid and aspartic acid residues, respectively. The post-translational formation of specific gamma-carboxyglutamic acid residues in protein C requires vitamin K. These unusual amino acid residues bind to calcium ions and are believed to be responsible for the interaction of the protein with phospholipid, which is required for the biological activity of protein C.
In contrast to the coagulation-promoting action of other vitamin K-dependent plasma proteins, such as factor VII, factor IX, and factor X, activated protein C (APC) acts as a regulator of the coagulation process through the inactivation of factor Va and factor VIIIa by limited proteolysis. The inactivation of factors Va and VIIIa by protein C is dependent upon the presence of acidic phospholipids and calcium ions. Protein S has been reported to regulate this activity by accelerating the APC-catalyzed proteolysis of factor Va (Walker, J. Biol. Chem. 255:5521-5524, 1980).
Protein C has also been implicated in the action of tissue-type plasminogen activator (Kisiel and Fujikawa, Behring Inst. Mitt. 73:29-42, 1983). Infusion of bovine APC into dogs results in increased plasminogen activator activity (Comp and Esmon, J. Clin. Invest. 68:1221-1228, 1981). Other studies (Sakata et al., Proc. Natl. Acad. Sci. USA 82:1121-1125, 1985) have shown that addition of APC to cultured endothelial cells leads to a rapid, dose-dependent increase in fibrinolytic activity in the conditioned media, reflecting increases in the activity of both urokinase-related and tissue-type plasminogen activators. APC treatment also results in a dose-dependent decrease in anti-activator activity.
In some parts of the world, it is estimated that approximately 1 in 16,000 individuals exhibit protein C deficiency. Protein C deficiency is associated with recurrent thrombotic disease (Broekmans et al., New Eng. J. Med. 309:340-344, 1983 and Seligsohn et al., New Eng. J. Med. 310:559-562, 1984) and may result from genetic disorders or from trauma, such as injury, liver disease or surgery. Protein C deficiency is generally treated with oral anticoagulants. Beneficial effects have also been obtained through the infusion of protein C-containing normal plasma (see Gardiner and Griffin in Brown, Grune & Stratton, eds., Prog. in Hematology, 13:265-278, 1983, New York). In addition, protein C is useful in treating thrombotic disorders, such as venous thrombosis (Smith et al., PCT Publication No. WO 85/00521)
Activated protein C may be preferred over the zymogen for the treatment of thrombosis. The use of activated protein C bypasses the need for in vivo activation of protein C, thus providing a faster acting therapeutic agent.
Finally, exogenous activated protein C has been shown to prevent the coagulopathic and lethal effects of gram negative septicemia (Taylor et al., J. Clin. Invest. 79:918-925, 1987). Data obtained from studies with baboons suggest that activated protein C plays a natural role in protecting against septicemia.
While protein C may be purified from clotting factor concentrates (Marlar et al., Blood 59:1067-1072, 1982) or from plasma (Kisiel, J. Clin. Invest. 64:761-769, 1979) and activated in vitro, it is a complex and expensive process, in part due to the limited availability of the starting material and the low concentration of protein C in plasma. Furthermore, the therapeutic use of products derived from human blood carries the risk of disease transmission by, for example, hepatitis virus, cytomegalovirus, or human immunodeficiency virus (HIV). For these reasons, it is preferable to produce human protein C and human activated protein C by genetic engineering techniques. In view of the clinical applicability of human protein C and human activated protein C in the treatment of thrombotic disorders, the production of useful quantities of human protein C and human activated protein C by recombinant DNA techniques is clearly invaluable.